DESCRIPTION: Formation of a heterogeneous population of cells is critical to embryogenesis and normal development; cell heterogeneity gives rise to different types of tissues and is also implicated in the onset and progression of diseases. Understanding cell heterogeneity holds important implications in human health, but requires specialized approaches, such as mass spectrometry (MS), that deliver high detection selectivity and sensitivity. A substantial portion o bioanalytical methodologies including single-cell MS, however, work ex vivo or rely on long-term cell cultures. These conditions potentially change the proteomic and, especially, the metabolomic composition and function of cells and complicate the interpretation of results on cell-to-cell differences. Herein we propose to introduce an in situ single-cell analysis platform based on MS and uncover metabolomic and proteomic differences among single cell that form in the actual, live, freely developing embryo of the South African clawed frog (Xenopus laevis) and the zebrafish (Danio rerio), both of which are well-established vertebrate models in cell and developmental biology and human disease research. Key aspects of this platform are in situ and high-throughput operation to identify and measure any given cell of interest directly in the specimen, a capability for repeated measurement of cell morphology and biochemical composition, label-free identification of diverse types of metabolites and peptides without having to know their presence before experiments, and scalability to different cell dimensions; thus, culturing and isolation of single cells are avoided. The platform is validated using single cells i the 16- and 32-cell Xenopus embryo that have highly reproducible tissue fates and exhibit known transcriptomic cell heterogeneity both in the horizontal and vertical body plan. Metabolites and peptides are measured in strategically selected identified cells along the dorso- ventral and animal-vegetal axes, and the resulting complex chemical information is mathematically evaluated to uncover similarity between cells that have the same identity in different embryos. Furthermore, we propose to extend single-cell investigations to the 1- to 16-cell zebrafish embryo, where cells are inherently smaller and cell heterogeneity is less understood during early development. Besides developing a new technology, the anticipated results provide new information on the spatiotemporal dynamics of cell heterogeneity in the actual developing embryo, providing important biochemical data for cell and developmental biology and human health. These outcomes are matched well with the goals of RFA-RM-13-021, Exceptionally Innovative Tools and Technologies for Single Cell Analysis. The proposed approach is adaptable to different physical and temporal resolutions, broad types of biomolecules, and different model systems to aid health research and the development of next-generation pharmaceuticals.